D'Ercole Claudia, de Marco Ario
Laboratory of Environmental and Life Sciences, University of Nova Gorica, Rožna Dolina, Nova Gorica, Slovenia.
Methods Mol Biol. 2023;2681:33-45. doi: 10.1007/978-1-0716-3279-6_3.
Phage display is an effective method to retrieve binders specific for a target epitope from a large clone library. Nevertheless, the panning process allows for the accumulation of some contaminant clones into the selected phage pool and, consequently, each clone requires individual screening to verify its actual specificity. This step is time-consuming, independently on the chosen method, and relies on the availability of reliable reagents. Since phages display a single binder responsible for the antigen recognition but their coat is formed by several repeats of the same proteins, the targeting of coat epitopes is often exploited to amplify the signal. Commercial anti-M13 antibodies are commonly labeled with peroxidase or FITC but customized antibodies might be necessary for specific applications. Here, we report a protocol describing the selection of anti-protoplast Adhirons that relies on the availability of nanobodies fused to a fluorescent protein to use during flow cytometry screening. Specifically, when preparing our Adhiron synthetic library, we designed a new phagemid that allows the expression of the clones fused to three tags. These can interact with a large variety of commercial and home-made reagents, selected according to the needs of the downstream characterization process. In the described case, we combined the ALFA-tagged Adhirons with an anti-ALFAtag nanobody fused with the fluorescent protein mRuby3.
噬菌体展示是从大型克隆文库中筛选出针对目标表位的特异性结合分子的有效方法。然而，淘选过程会使一些污染性克隆积累到选定的噬菌体库中，因此，每个克隆都需要单独筛选以验证其实际特异性。这一步骤无论采用何种方法都很耗时，且依赖于可靠试剂的可用性。由于噬菌体展示的是负责抗原识别的单一结合分子，但其衣壳由相同蛋白质的多个重复序列组成，因此常常利用针对衣壳表位的靶向作用来放大信号。商业抗M13抗体通常用辣根过氧化物酶或异硫氰酸荧光素标记，但特定应用可能需要定制抗体。在此，我们报告一种方案，该方案描述了抗原生质体Adhirons的筛选，其依赖于与荧光蛋白融合的纳米抗体的可用性，以便在流式细胞术筛选中使用。具体而言，在制备我们的Adhiron合成文库时，我们设计了一种新的噬菌粒，它允许表达与三个标签融合的克隆。这些标签可以与根据下游表征过程的需要选择的多种商业和自制试剂相互作用。在所述案例中，我们将带有ALFA标签的Adhirons与与荧光蛋白mRuby3融合的抗ALFA标签纳米抗体相结合。
